The overall objectives remain to understand and define the cellular mechanisms culminating in liver cell cytotoxicity. Nonalcoholic fatty liver disease (NAFLD), present in up to 30% of the American population, is characterized by high levels of circulating free fatty acids (FFA) and hepatocyte apoptotosis. We and others have demonstrated that FFA directly induce hepatocyte apoptosis, termed lipoapoptosis. Due to the public health relevance of liver lipoapoptosis, our program is currently focused on the cellular and subcellular mechanisms of FFA-mediated liver injury. Specifically, our long term objectives are to understand the pivotal mechanisms involved in FFA-induced hepatocyte apoptosis. Based on extensive preliminary data, we propose the novel CENTRAL HYPOTHESIS that FFA signal lipoapoptosis by regulating expression and function of specific Bcl-2 (B-cell lymphoma-2) proteins. We will now employ current and complementary, molecular, biochemical and cell biological approaches to ascertain how FFA trigger this largely unexplored pathway of apoptosis. Our SPECIFIC AIMS will test three hypotheses. FIRST, we will directly test the hypothesis that FFA upregulate expression of PUMA (p53 upregulated modifier of apoptosis), a proapoptotic Bcl-2 protein: a) by a c-Jun-N-terminal Kinase (JNK)-driven activation of the transcription factor activator protein 1 (AP1) complex; and b) PUMA expression is essential for activation of the pro-apoptotic Bcl-2 proteins, Bax and/or Bak. SECOND, we will test the hypothesis that FFA-mediate degradation of Mcl-1 (myeloid cell leukemia-1), a potent antiapoptotic Bcl-2 protein: a) by a PKC8 (protein kinase C 8)-dependent proteasome degradation pathway; and b) is essential for rapid induction of lipoapoptosis. FINALLY, we will test the hypothesis in animal models of nonalcoholic fatty liver disease (NAFLD) that hepatocyte apoptosis and liver injury: a) are reduced by genetic deletion of PUMA; and b) are exacerbated by genetic deletion of Mcl-1 and attenuated by its overexpression. The proposal is technically and conceptually innovative as it tests new mechanisms for FFA cytotoxicity using a variety of sophisticated technologies. Our results will yield new mechanistic insights into lipoapoptosis, further clarify the molecular pathogenesis of NAFLD, and identify potential strategies for the treatment of NAFLD. PUBLIC HEALTH RELEVANCE: The grant examines the cellular mechanisms by which serum free fatty acids, which are elevated in diabetes and obesity syndromes, cause liver injury. We propose that these free fatty acids increase liver cell expression of pro death proteins and deplete the cell of survival factors. This imbalance causes liver cell death which in turn induces liver inflammation and cirrhosis. The results of these studies are germane to mechanisms of liver injury in the common syndrome of nonalcoholic fatty liver disease, and have the potential to identify new therapeutic strategies for this liver disease.